Canonical Wnt signaling is initiated by binding of Wnt proteins to members of the Frizzled family and subsequent complex formation with lipoprotein receptor-related proteins 5/6 (LRP5/6). Here, we show that LRP6 is palmitoylated on a juxtamembranous cysteine and that palmitoylation is required for exit from the endoplasmic reticulum (ER). We propose that palmitoylation serves to tilt the long, 23-residue transmembrane domain of LRP6 with respect to the plane of membrane to prevent a hydrophobic mismatch and subsequent recognition by the ER quality control. In support of this model, a palmitoylation-deficient LRP6 mutant could be rescued from ER retention by deletion of two to four residues in the transmembrane domain. Importantly, we found that palmitoylation-deficient LRP6 was retained in the ER by a completely novel monoubiquitinationdependent ER retention mechanism. Mutation of a specific lysine indeed abolished ubiquitination of palmitoylation-deficient LRP6 and led to a rescue from ER retention. Finally, at the cell surface, we found that interplay between palmitoylation and ubiquitination was necessary for efficient Wnt signaling.he signaling of Wnt plays a critical role in a variety of developmental and adult processes in all metazoan and in diseases such as cancer (1). Canonical Wnt signaling via ␤-catenin is transduced by two receptor families: the Frizzled proteins and lipoprotein receptor-related proteins (LRPs) 5 and 6. Wnt binds to Frizzled, which then associates with LRP5/6. The cytoplasmic domains of LRP5/6, upon receptor activation by Wnt proteins, recruit the cytosolic scaffold protein axin to the membrane, displacing it from the so-called destruction complex (containing axin, glycogen synthase kinase 3␤, Wilms tumor suppressor, and ␤-catenin) involved in the degradation of ␤-catenin by the proteasome (for review see ref.2). This rescue of ␤-catenin allows it to translocate into the nucleus and interact with T cell factor (TCF)/lymphoid enhancer factor to activate transcription (3). As a result, Wnt activates multiple intracellular cascades, leading to cell differentiation, proliferation, migration, and polarity.LRP6 is a type I membrane protein with a large, 1,351-aa extracellular domain, containing multiple ␤-propeller and EGF domains, and a 220-aa-long cytoplasmic tail. As all type I transmembrane proteins, LRP6 is synthesized with an Nterminal signal sequence that targets it to the endoplasmic reticulum (ER). Folding of the LRP6 ␤-propeller domains in the lumen of the ER requires the help of an ER-resident chaperone called mesoderm development candidate 2 (Mesd) in mammals and Boca in Drosophila (4, 5). In the absence of Mesd, LRP6 is retained in the ER and thus fails to reach the cell surface.Human LRP6 contains six cysteine residues in its cytoplasmic tail, which are potential sites for palmitoylation (6). This lipid can serve to tether soluble proteins to membranes but is also found attached to the cytoplasmic domains of membrane proteins often close to the transmembrane region (7). The f...